Skeletal Removal of Bisphosphonates

ABSTRACT

Disclosed herein are methods and compositions for removing or displacing bisphosphonates in skeletal tissue.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under R01DE022552, SBIR1R43DE025524-01, and SBIR 2R44DE025524-02 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to methods of treating osteonecrosis andother skeletal disorders and symptoms, including Bisphosphonate RelatedOsteonecrosis of the Jaw (BRONJ).

2. Description of the Related Art

Osteonecrosis of the jaw (ONJ) is a rare but severe oral complication,which is characterized by symptoms consisting painful bone exposure orfistulation that do not resolve over several months to years. It wasinitially described as Bisphosphonate Related ONJ (BRONJ), whenidentified in patients treated with potent antiresorptive bisphosphonate(BP) drugs, particularly nitrogen-containing BPs (N-BPs) such aszoledronate (ZOL), pamidronate, and alendronate, and was also foundlater in some patients taking other antiresorptive or antiangiogenicmedications, such as denosumab. In some cases, ablation surgery ofnecrotic oral and maxillofacial bones has been required, significantlyaffecting patients' life quality, however, current preventive andtreatment modalities are limited. Clinical reports and patient surveyshave indicated that dental procedures such as routine tooth extractionand denture wearing markedly increase the risk of developing ONJ, thuscausing uncertainty and apprehension among dental healthcareprofessionals and patients in recent years.

The updated position paper released by American Association of Oral andMaxillofacial Surgeons (AAOMS) redefined ONJ as Medication RelatedOsteonecrosis of the Jaw (MRONJ), expanding the scope of its previousposition paper on BRONJ. MRONJ reflects the inclusion of ONJ resultingfrom or associated with the administration of medications in addition toactive BPs, for example, receptor activator of nuclear factor kappa-Bligand (RANKL) inhibitors (e.g., denosumab) and antiangiogenic therapiesas possible associated agents.

The mechanisms of action of active BPs and denosumab are considered bythose skilled in the art to be different. The pharmacological mechanismof active BPs is based on their resemblance to pyrophosphate. Because ofa high affinity for calcium ions in the bone, active BPs accumulate onthe bone and targets osteoclasts. During bone resorption, active BPs areendocytosed by osteoclasts, and inhibit an important enzyme, farnesylpyrophosphate synthase, in the mevalonate pathway. As a result, theactivity of osteoclasts is decreased, leading to the reduction of boneloss. By contrast, denosumab inhibits RANKL, a protein that binds to theRANK receptors on the pre-osteoclasts. Denosumab prevents RANKL frombinding to RANK receptors, thus impairing osteoclast formation.

A “drug holiday” has been recommended by AAOMS as a possible preventivemeasure for the patients and has shown some promising results inpatients with denosumab therapy; however, its effectiveness for patientswho have been treated with active BPs has not been well established,which may partially due to their different mechanisms of actions and theprolonged half-lives of active BPs in bone. After administration, asmuch as about 50% of an active BP is incorporated into bone and the restis rapidly excreted through the kidneys. There is no systemic metabolismof active BPs, contributing to their prolonged half-lives, which mayreach several months to over 10 years depending on the given BP. Infact, the US FDA determined that there was “no substantial dataavailable to guide decisions regarding the initiation or duration of adrug holiday”.

Because active BPs have been marketed for nearly 15 years, there arenumerous patients who have been treated with active BPs and may have theactive BPs retained in their skeletal systems. Therefore, BRONJcontinues to present a healthcare threat to many who were and aretreated with active BPs.

Thus, a need exists for methods of treating, preventing, and/orinhibiting BRONJ, as well as other bone and skeletal issues caused by orassociated with treatment with active BPs.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides methods for removingor displacing an active BP in a skeletal tissue, such as bone tissue,which comprise administering to the skeletal tissue one or more inactiveBPs. In some embodiments, the active BP in the skeletal tissue, such asbone tissue, is removed or displaced in vivo in a subject. In someembodiments, the present invention provides methods for treating,reducing, preventing, or inhibiting BRONJ and/or abisphosphonate-related symptom in a subject, which comprisesadministering to the subject one or more inactive BPs. In someembodiments, the present invention is directed to use of one or moreinactive BPs for removing or displacing an active BP in a skeletaltissue, such as bone tissue. In some embodiments, the present inventionis directed to a medicament for the treatment of BRONJ and/or abisphosphonate-related symptom, which comprises a therapeuticallyeffective amount of one or more inactive BPs.

Examples of inactive BPs suitable for use in the various embodiments ofthe present invention include those described at paragraphs [0044] to[0050] and pharmaceutically acceptable salts, solvates, and prodrugsthereof and examples of active BPs include those described at paragraph[0043]. In some embodiments, the active BP being removed or displaced isan active nitrogen-containing bisphosphonate. In some embodiments, theactive nitrogen-containing bisphosphonate is alendronate, ibandronate,minodronate, pamidronate, risedronate, or zoledronate. In someembodiments, the one or more inactive BPs is a low activitybisphosphonate. In some embodiments, the one or more inactive BPs lackan α-hydroxy group, have a pyridyl side chain that is para-substituted,and/or comprise a bisphosphonate covalently attached to a fluorescentcompound. In some embodiments, the active BP being removed or displacedis an active nitrogen-containing bisphosphonate and the one or moreinactive BPs is a low activity bisphosphonate. In some embodiments, theactive nitrogen-containing bisphosphonate is alendronate, ibandronate,minodronate, pamidronate, risedronate, or zoledronate and the one ormore inactive BPs is a low activity bisphosphonate. In some embodiments,the active nitrogen-containing bisphosphonate is alendronate,ibandronate, minodronate, pamidronate, risedronate, or zoledronate andthe one or more inactive BPs lack an α-hydroxy group. In someembodiments, the active nitrogen-containing bisphosphonate isalendronate, ibandronate, minodronate, pamidronate, risedronate, orzoledronate and the one or more inactive BPs have a pyridyl side chainthat is para-substituted. In some embodiments, the activenitrogen-containing bisphosphonate is alendronate, ibandronate,minodronate, pamidronate, risedronate, or zoledronate and the one ormore inactive BPs lack an α-hydroxy group and/or have a pyridyl sidechain that is para-substituted. In some embodiments, the activenitrogen-containing bisphosphonate is alendronate, ibandronate,minodronate, pamidronate, risedronate, or zoledronate and the one ormore inactive BPs comprise a bisphosphonate covalently attached to afluorescent compound. In embodiments where the mode of administration issystemic administration, the one or more inactive BPs is not etidronate.In some embodiments, the one or more inactive BPs has the followingstructure:

wherein X is H, hydroxyl, amino, halo, alkyl, or aryl; Y is hydroxyl,amino, alkyl, aryl, or heterocycle; and Z is a detectable label, such asa fluorophore, that may be present or absent. In some embodiments, X isa hydroxyl or H and Y is a pyridyl group that is para-substituted. Insome embodiments, Z is present. In some embodiments, Z is absent.

In some embodiments of the methods of the present invention, the one ormore inactive BPs is systemically administered to a subject, with theproviso that the one or more inactive BPs is not etidronate. In someembodiments, the one or more inactive BPs is locally administered to asite where the active BP is to be removed or displaced. In someembodiments, the one or more inactive BPs is administered orally to thesubject. In some embodiments, the one or more inactive BPs isadministered to a gingival tissue and/or a palatal tissue of thesubject. In some embodiments, the one or more inactive BPs isadministered by injection at the site where active BP is to be removedor displaced. In some embodiments, the one or more inactive BPs isadministered topically at the site where active BP is to be removed ordisplaced. In some embodiments, the one or more inactive BPs isadministered by intraoral application to the site of a dentoalveolarprocedure performed on the subject. In some embodiments, the one or moreinactive BPs is administered before, during, and/or after thedentoalveolar procedure. In some embodiments, the one or more inactiveBPs is administered by direct injection into the mucosa at or near thesite of a dentoalveolar procedure. In embodiments where the one or moreinactive BPs being administered is etidronate, the mode ofadministration is local administration. In some embodiments, the one ormore inactive BPs is administered as a pharmaceutical composition. Insome embodiments, the one or more inactive BPs is provided in the formof a pharmaceutical composition. In some embodiments, the pharmaceuticalcomposition is a composition or formulation as described at paragraphs[0064] to [0067]. In some embodiments, the pharmaceutical composition isa deformable nanovesicles formulation. In some embodiments, thepharmaceutical composition is a phospholipid-based deformablenanovesicles formulation. In some embodiments, an effective amount ofthe one or more inactive BPs is administered. In some embodiments, atherapeutically effective amount of the one or more inactive BPs isadministered to the subject. In some embodiments, the subject who isadministered the one or more inactive BPs has been treated with anactive BP. In some embodiments, the subject who is administered the oneor more inactive BPs is being treated with an active BP. In someembodiments, the subject who is administered the one or more inactiveBPs will be treated with an active BP.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are intended toprovide further explanation of the invention as claimed. Theaccompanying drawings are included to provide a further understanding ofthe invention and are incorporated in and constitute part of thisspecification, illustrate several embodiments of the invention, andtogether with the description explain the principles of the invention.

DESCRIPTION OF THE DRAWINGS

This invention is further understood by reference to the drawingswherein:

FIG. 1, Panels A-C, illustrates displacement of an adsorbedfluorescently labeled BP (FL-BP) from a hydroxyapatite surface mimickingbone by a different fluorescently labeled BP. Synthetic bone/dentine(calcium phosphate, CaP)-coated culture wells were pre-treated witheither ROX-RIS or 5-FAM-RIS (50 μM). After challenged by seriallydiluted doses of 5-FAM-RIS or ROX-RIS (from 0 to 50 μM), respectively,Carboxyl-X-Rhodamine (ROX) and carboxyfluorescein (FAM) fluorescenceintensities of the CaP-coated wells were measured. Panel A: ROX-RIS inCaP (first bars of each set) remained near the pre-adsorbed 50 μM leveluntil 5 μM or greater concentrations of 5-FAM-RIS challenged. Incontrast, 5-FAM-RIS (second bars of each set) was rapidly displaced byconcentrations of ROX-RIS as low as 1 μM. Panel B: Pre-adsorbed FAM-RISwas replaced by ROX-RIS between 10 μM to 50 μM. Panel C: Thequantitative measurement of FAM and ROX fluorescent signals wasconducted by standardized fluorescent biophotonics and a proprietaryprogram (LAS3000, FUJIFILM, Tokyo, Japan).

FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D schematically show the synthesisof various FL-BPs.

FIG. 3 are images of fluorescently labeled risedronate (RIS) adsorbed ondentine surface (green spots within circle in top image, and green spotsand line underneath semi-circle in bottom image). An osteoclast (bluecircle in top image and semi-circle in bottom image) adhering to thesurface can be seen to ingest the released drug as it forms a localresorption pit (dashed white line). Upper image: 1 μm×y image 8 μm abovethe surface of the dentine; lower image: zx image of the sameosteoclast.

FIG. 4 shows the structure of RIS,2-(pyridin-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), and1-hydroxy-2-(pyridin-4-yl)ethane-1,1-diylbis(phosphonic acid) (p-RIS).

FIG. 5A and FIG. 5B schematically show the synthesis of some FL-BPs.

FIG. 6 shows the structural formulas of several FL-BPs.

FIG. 7 is a bar graph showing hydroxyapatite (HAP) binding affinity ofZoledronate (ZOL), ROX-RIS, 5-FAM-ZOL, and AF647-ZOL. Relative retentiontimes were normalized to RIS.

FIG. 8A to FIG. 8D show adsorption isotherms for binding of four FL-BPsto hydroxyapatite (HAP) at pH 6.8 (top graphs) with Scatchard plots ofthe same data (bottom graphs), data are mean±SD (n=3). FIG. 8A shows theadsorption isotherm for 5(6)-ROX-RIS, FIG. 8B shows the adsorptionisotherm for 5(6)-ROX-RISPC, FIG. 8C shows the adsorption isotherm forAF647-RIS, and FIG. 8D shows the adsorption isotherm for AF647-RISPC.

FIG. 9 shows emission wavelengths of 3 different FL-BPs (ROX-RIS,AF647-ZOL and 5-FAM-ZOL). From left to right, the curves are 5-FAM-ZOL,ROX-RIS, and AF647-ZOL. Overlap between 5-FAM and AF647 is minimal.

FIG. 10 are representative images of mice maxilla. AF647-ZOL was orallyinjected into maxilla of mice pre-injected with 5-FAM-ZOL. A dosedependent adsorption of AF647-ZOL was observed at and around theinjection site (arrow). Reduction of FAM-ZOL signal was observedqualitatively (dotted line). Fluorescent imaging of the femur showedthat, unlike intravenous administration, intra-oral injection ofAF647-ZOL did not result in systemic distribution to the distantskeletal system.

FIG. 11 shows fluorescent biophotonic images of mouse maxilla after5-FAM-ZOL IV injection (left panel) or ROX-RIS intraoral injection(center and right panels).

FIG. 12A to FIG. 12C show the displacement and replacement of AF647-ZOLand 5-FAM-ZOL. FIG. 12A are fluorescent images of cranial bone.AF647-ZOL (50 μM) was pre-adsorbed on cranial bone via IV injection.5-FAM-ZOL at the indicated doses was directly injected to thesub-periosteal space. FIG. 12B is a graph showing that the 5-FAM-ZOLsignal increased with increasing injection dose, while the AF647-ZOLsignal decreased. The first bars of each set are for AF647-ZOL and thesecond bars of each set are for 5-FAM-ZOL. *, P<0.05 vs Veh by Dunnett'stest. FIG. 12C are images of cranial bone cryo-cross sections, whichrevealed localized displacement of AF647-ZOL and replacement by5-FAM-ZOL.

FIG. 13 shows the results of micro CT evaluation of mouse femurtrabecular structure. The pharmacological function of 5-FAM-ZOL wasexamined in vitamin D deficient (VitD (−)) rats (n=3 in each group).5-FAM-ZOL effectively blocked catabolic bone remodeling resulting inincreased trabecular bone structure. Bars are 1.0 mm. *: p<0.05 byStudent's t-test.

FIG. 14 shows the results of micro CT evaluation of mouse femurtrabecular structure. IV injection of ZOL, but not AF647-ZOL,significantly increased bone structural parameters.

FIG. 15 are images showing that the mouse ONJ model is a suitable BRONJmodel. Healing of the tooth extraction wound was delayed in theZOL-injected mice (arrowhead). Oral epithelial hyperplasia (arrows)resulted in the exposure of necrotic alveolar bone (red dotted line,substantially horizontal, in bottom right panel).

FIG. 16A to FIG. 16C demonstrate the prevention or inhibition of BRONJin the BRONJ mouse model. FIG. 16A schematically shows the mouse modelstudies involving ZOL IV injection followed by tooth extraction.AF647-ZOL was applied by intra-oral injection or IV injection 1 dayprior to tooth extraction. FIG. 16B are images of mouse maxilla and theoral mucosa inflammation and swelling (white dotted lines, left andright panels) with various degrees of jawbone exposure (arrow) at thetooth extraction site of ZOL-treated mice (BRONJ control: n−4). In theAF647-ZOL intra-oral injection group (n=4) (middle panel), all miceexhibited excellent tooth extraction wound healing without chronicinflammation or swelling. The AF647-ZOL IV injection group (n=4) (rightpanel) showed attenuated BRONJ-like lesions. FIG. 16C presentsstandardized fluorescent biophotonics images of mouse femurs showingthat the AF647-ZOL signal could be clearly observed in femurs afterAF647-ZOL IV injection. By contrast, intra-oral injection did not resultin a detectable fluorescent signal in femurs.

FIG. 17 shows the structural formulas of a several alkylidenebisphosphonates, including methylene hydroxyl bisphosphonate (MHDP),ethylene hydroxyl bisphosphonate (EHDP), methylene bisphosphonate (MBP),clodronate, tiludronate, 2-(pyridin-4-yl)ethane-1,1-diyl bisphosphonicacid (p-PyrEBP), and 1-hydroxy-2-(pyridin-4-yl)ethane-1,1-diylbis(phosphonic acid) (p-RIS).

FIG. 18A1 to FIG. 18A4 schematically present the preparation of ICGanalogs for the synthesis of near infrared FL-BPs (ICG-BPs) that can beused for displacement of active BPs. FIG. 18A1 shows a general schematicand synthetic Route A, FIG. 18A2 shows synthetic Route B, FIG. 18A3synthetic Route C, and FIG. 18A4 shows synthetic Route D. FIG. 18Bpresents the structures of several such ICG-BPs.

FIG. 19A to FIG. 19C present evidence that certain alkylidenebisphosphonates can be used to displace active BPs in vivo in the mousemodel. FIG. 19A shows the results of micro CT evaluation of mouse femurtrabecular structure demonstrating the lack of anti-resorptive effect ofMHDP, which remained at the baseline level as the saline (0.9% NaCl)vehicle group. Zoledronate (ZOL) exhibited a significant anti-resorptiveeffect as shown in increased bone volume over tissue volume (BV/TV) andby decreased connectivity density (ConnD) in trabecular bone. FIG. 19Boutlines the protocol for the experiments using MHDP to treat C57Bl6mice. An ONJ-like lesion was generated by ZOL IV injection into themouse, followed by extraction of the maxillary first molar. One dayprior to the tooth extraction, a group of mice received intra-oralinjection of 2 μg MHDP and another group received IV injection of 100 μgMHDP. FIG. 19C presents images of the mouse maxilla. The left panelshows that ZOL-injected mice without the MHDP treatment developedONJ-like lesions: extensive inflammation of oral mucosa and exposedjawbone. Intra-oral injection (middle panel) and IV injection (rightpanel) of MHDP prevented the development of ONJ-like lesions.

FIG. 20 shows representative histology of mouse maxilla at the toothextraction site. ZOL-injected mice demonstrated the development of anONJ-like lesion 2 weeks after maxillary molar extraction, highlighted byan exposed wound (Exp) flanked by oral epithelial hyperplasia (arrows).The tooth extraction socket (black dotted line) did not show bone woundhealing and the surface of palatal bone (red dotted line) was non-vital(osteonecrosis), which interfaced to the localized and intenseinflammatory reaction (arrowheads). ZOL-mice treated with the one timeoral injection of AF647-ZOL or MHDP did not develop an ONJ-like lesion.The tooth extraction socket (black dotted line) was filled withregenerating bone and oral mucosa inflammation was much attenuated. Noosteonecrosis of the maxillary bone was observed.

FIG. 21, Panels A and B, show ONJ prevention by intra-oral injection ofinactive BPs in mice. Panel A is a diagram of the experimental timeline. Mice treated by IV injection of ZOL (500 μg/kg) received anintra-oral injection of AF647-ZOL, MHDP or ETI (etidronate) one dayprior to maxillary 1^(st) molar extraction. The development of ONJ wasevaluated 14 days after tooth extraction. Panel B shows the diseasecontrol mice (ZOL-NaCl) developed ONJ-like lesions at the toothextraction sites, which appeared to be prevented by the intra-oralinjection of AF647-ZOL, MHDP or ETI.

DETAILED DESCRIPTION OF THE INVENTION

Medication-related osteonecrosis of the jaw (MRONJ) is clinicallycharacterized as unresolved exposure of or fistula communication topartially necrotic jawbone in the oral cavity and is frequentlyassociated with dentoalveolar procedures. Among the antiresorptive andantiangiogenic medications, treatment with antiresorptivebisphosphonates is prominently associated with reported clinical casesof MRONJ. Repeated administration of antiresorptive bisphosphonatesresult in high accumulation levels of the antiresorptive bisphosphonatesin bone that are often associated with osteonecrosis of the jaw (ONJ).Thus, ONJ in subjects who have been or are being treated with active BPsis generally referred to as Bisphosphonate Related Osteonecrosis of theJaw (BRONJ).

As used herein, “active bisphosphonates (active BPs)” refer tobisphosphonates that exhibit potent antiresorptive activity and are usedin antiresorptive therapies. As used herein “bisphosphonates” generallyrefer to compounds that have two phosphonate groups covalently linked toa carbon. Active bisphosphonates include “active nitrogen-containingbisphosphonates (active N-BPs)”, which refers to bisphosphonates thathave a chemical structure containing a nitrogen that forms hydrogenbonds with Thr201 and the carbonyl of Lys200 of human farnesyldiphosphate synthase (FPPS). Examples of active N-BPs includealendronate, ibandronate, minodronate, pamidronate, risedronate, andzoledronate.

As used herein, “inactive bisphosphonates (inactive BPs)” refer tobisphosphonates that are completely inactive as antiresorptive agents orpartially inactive as antiresorptive agents as compared to active BPsthat have been and/or are used in antiresorptive therapies. Partiallyinactive BPs are referred to herein as “low activity bisphosphonates(low activity BPs)”. In other words, inactive BPs include low activityBPs. In some embodiments, inactive BPs have a chemical structure thatlacks an α-hydroxy group. In some embodiments, inactive BPs have achemical structure that has a pyridyl side chain that ispara-substituted. In some embodiments, inactive BPs lack an α-hydroxygroup and have a pyridyl side chain that is para-substituted. In someembodiments, the one or more inactive BPs has the following structure:

wherein X is H, hydroxyl, amino, halo, alkyl, or aryl; Y is hydroxyl,amino, alkyl, aryl, or heterocycle; and Z is a detectable label, such asa fluorophore, that may be present or absent. In some embodiments, X isa hydroxyl or H and Y is a pyridyl group that is para-substituted. Insome embodiments, Z is present. In some embodiments, Z is absent.

Inactive BPs, according to the present invention, exhibit bindingaffinities for hydroxyapatite (HAP). In some embodiments, inactive BPsexhibit a binding affinity for hydroxyapatite that is more than that ofactive BPs. In some embodiments, inactive BPs competitively inhibit thebinding of active BPs to hydroxyapatite. In some embodiments, inactiveBPs displace active BPs that are bound to hydroxyapatite whenadministered thereto. In some embodiments, inactive BPs exhibit a boneaffinity that is the same, or substantially similar to active N-BPs andlittle to no anti-osteoclastic activity. In some embodiments, inactiveBPs exhibit a bone affinity that is higher than active N-BPs and littleor none of the anti-osteoclastic activity of the active N-BPs.

In some embodiments, an inactive BP may contain a nitrogen within itschemical structure so long as the inactive BP inhibits human farnesyldiphosphate synthase (FPPS) to a lesser degree than the active N-BPbeing displaced or replaced. In some embodiments, an inactive BP maycontain a nitrogen within its chemical structure so long as the strengthof any hydrogen bonds formed between the nitrogen with Thr201 and withthe carbonyl of Lys200 of human farnesyl diphosphate synthase (FPPS) isless than that of the hydrogen bonds formed between the active N-BPbeing displaced or replaced and Thr201 and the carbonyl of Lys200 ofFPPS. In some embodiments, an inactive BP may contain a nitrogen withinits chemical structure so long as the nitrogen does not form hydrogenbonds with Thr201 and the carbonyl of Lys200 of human farnesyldiphosphate synthase (FPPS). Enzymatic binding activity assays andprotein modeling methods in the art may be used to screen forbisphosphonates that do not have a nitrogen that forms hydrogen bondswith Thr201 and the carbonyl of Lys200 of human farnesyl diphosphatesynthase (FPPS). See, e.g., Ebetino, et al. (2011) Bone 49(1): 20-33;and Kavanagh, et al. (2006) PNAS, 103(20): 7829-7834. In someembodiments, inactive BPs have an inhibitory IC₅₀ of human farnesyldiphosphate synthase (FPPS), as measured according to the procedure inDunford, et al. (2008) J Medicinal Chemistry 51(7): 2187-2195, of morethan 4.1 nM, preferably 10 nM or more, more preferably 100 nM or more,even more preferably 500 nM or more, and most preferably 1000 nM ormore. In some embodiments, inactive BPs do not inhibit proteinprenylation at concentrations below 100 μM, as measured according to theprocedure in Sun, et al. (2016) Bioconjugate Chem 27(2): 329-340. Insome embodiments, inactive BPs do not inhibit bone resorption in in vivomodels of bone metabolism, such as the Schenk or growing rat model(Seitsema, et al. (1989) Drugs Exptl. Clin. Res. XV(9): 389-396) atconcentrations below those of active BPs. Examples of inactive BPsinclude 2-(pyridin-4-yl)ethane-1,1-diyl bisphosphonic acid (p-PyrEBP),1-hydroxy-2-(pyridin-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS),methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate(MHDP), etidronate (EHDP), clodronate, isclodronate, tiludronate,2-hydroxy-2-phosphono-3-(pyridin-3-yl)propanoic acid (3-PEHPC), and2-hydroxy-3-(imidazo[1,2-a]pyridin-3-yl)-2-phosphonopropanoic acid(3-IPEHPC).

In some embodiments, inactive BPs have a detectable label, such as afluorescent compound, attached thereto. In some embodiments, theinactive BPs comprise a bisphosphonate (which may be an active BP or aninactive BP) covalently attached to a fluorescent compound, such as ROX,FAM, AF647, ICG, Cy5, Sulfo-Cy5, Cy7, and IRDye 800CW. Examples ofinactive BPs having a fluorescent compound conjugated thereto includecompounds 7a1-7f2 of FIG. 2A to FIG. 2D, fluorescent p-PyrEBP conjugatessuch as those of FIG. 5A, fluorescent p-RIS conjugates such as those ofFIG. 5B, the IGC compounds synthesized according to FIG. 18A1 to FIG.18A4, and the IGC compounds set forth in FIG. 18B. In some embodiments,the inactive BPs that have a fluorescent compound conjugated thereto are5-FAM-dRIS, 5(6)-FAM-dRIS, 5(6)-FAM-RIS, 5(6)-FAM-RISPC, 5(6)-RhR-RIS,5(6)-RhR-dRIS, 5(6)-RhR-RISPC, 5(6)-ROX-RIS, 5(6)-ROX-RISPC, 5-FAM-RIS,5-FAM-ZOL, 6-FAM-RIS, 800CW-ZOL, AF647-RIS, AF647-RISPC, AF647-ZOL,800CW-RIS, 800CW-ZOL, 800CW-RISPC, ICG-RIS, ICG-ZOL, ICG-RISPC, andICG-p-pyrEBP.

Inactive BPs also include pharmaceutically acceptable solvates, salts,and prodrugs of bisphosphonates, which are completely inactive asantiresorptive agents or partially inactive as antiresorptive agents ascompared to active BPs that have been and/or are used in antiresorptivetherapies. A “pharmaceutically acceptable solvate” refers to a solvateform of a specified compound that retains the biological activity, e.g.,the anti-resorptive activity (or lack thereof), of the given compound.Examples of solvates include compounds of the invention in combinationwith water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethylacetate, acetic acid, ethanolamine, or acetone. Those skilled in the artof organic chemistry will appreciate that many organic compounds canform complexes with solvents in which they are reacted or from whichthey are precipitated or crystallized. These complexes are known as“solvates”. For example, a complex with water is known as a “hydrate”.Many organic compounds can exist in more than one crystalline form. Forexample, crystalline form may vary from solvate to solvate. Thus, allcrystalline forms of inactive BPs and solvates thereof are within thescope of the present invention.

The term “pharmaceutically acceptable salts” refers to salt forms thatare pharmacologically acceptable and substantially non-toxic to thesubject being treated with the compound of the invention.Pharmaceutically acceptable salts include conventional acid-additionsalts or base-addition salts formed from suitable non-toxic organic orinorganic acids or inorganic bases. Exemplary acid-addition saltsinclude those derived from inorganic acids such as hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid,phosphoric acid, and nitric acid, and those derived from organic acidssuch as p-toluenesulfonic acid, methanesulfonic acid, ethane-disulfonicacid, isethionic acid, oxalic acid, p-bromophenylsulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, 2-acetoxybenzoic acid,acetic acid, phenylacetic acid, propionic acid, glycolic acid, stearicacid, lactic acid, malic acid, tartaric acid, ascorbic acid, maleicacid, hydroxymaleic acid, glutamic acid, salicylic acid, sulfanilicacid, and fumaric acid. Exemplary base-addition salts include thosederived from ammonium hydroxides (e.g., a quaternary ammonium hydroxidesuch as tetramethylammonium hydroxide), those derived from inorganicbases such as alkali or alkaline earth-metal (e.g., sodium, potassium,lithium, calcium, or magnesium) hydroxides, and those derived fromnon-toxic organic bases such as basic amino acids.

A “pharmaceutically acceptable prodrug” is a compound that may beconverted under physiological conditions or by solvolysis to thespecified compound or to a pharmaceutically acceptable salt of suchcompound. “A pharmaceutically active metabolite” refers to apharmacologically active product produced through metabolism in the bodyof a specified compound or salt thereof. Prodrugs and active metabolitesof a compound may be identified using techniques known in the art. See,e.g., Bertolini, G. et al., (1997) J. Med. Chem. 40:2011-2016; Shan, D.et al., J. Pharm. Sci., 86(7):765-767; Bagshawe K., (1995) Drug Dev.Res. 34:220-230; Bodor, N., (1984) Advances in Drug Res. 13:224-331;Bundgaard, H., Design of Prodrugs (Elsevier Press, 1985) and Larsen, I.K., Design and Application of Prodrugs, Drug Design and Development(Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991).

As disclosed herein, previously adsorbed active BPs in skeletal tissue,such as bone, can be displaced or removed by newly administered inactiveBPs. Therefore, in some embodiments the present invention is directed tomethods of removing or displacing bisphosphonates in skeletal tissue byadministering one or more inactive BPs to the skeletal tissue. In someembodiments, the present invention is directed to methods of treating,inhibiting, or reducing BRONJ and/or bisphosphonate-related symptoms byadministering to subjects in need thereof one or more inactive BPs. Asused herein, “bisphosphonate-related symptoms” include symptoms, such aspainful bone exposure and fistulation, abnormal skeletal fractures,etc., that are caused by or associated with treatment with an active BP.Examples of abnormal skeletal fractures in subjects who have beentreated with active N-BPs include atypical femoral fractures andfractures of the hip. See, e.g., Paziana, et al. (2011) Bone 43:103-110.

Locally administered inactive BPs are capable of binding to bone at theadministration site without being distributed systematically in asignificant concentration. In some embodiments, one or more inactive BPsare locally administered, e.g., injection into the bone tissue at thesite where the active BP is to be displaced or removed, topicaladministration on the site of the bone tissue which is exposed during,for example, a surgical procedure, or application (e.g., injection) inthe tissue adjacent to the site of the bone tissue to be treated in theform of a pharmaceutical formulation, e.g., on or in a drug deliveryparticle such as a liposome or nanovesicle, that reduces, inhibits, orprevents systemic distribution of the one or more inactive BPs. In someembodiments, the one or more inactive BPs are delivered in the form ofnanovesicles. In some embodiments, the nanovesicles are nondeformablenanovesicles. In some embodiments, the nanovesicles are deformablenanovesicles. See, e.g., WO 2017/087685 and Subbiah, et al. (2017) JDrug Delivery, Article ID 4759839. In some embodiments, one or moreinactive BPs are locally administered to subjects at the sites whereBRONJ and/or bisphosphonate-related symptoms are likely to develop inwho have been or are being treated with active BPs as a preventativemeasure against the development of BRONJ and/or bisphosphonate-relatedsymptoms.

In some embodiments, the subjects to be treated are ones who are notcandidates for a drug holiday. In some embodiments, the subject to betreated is one that suffers from osteoporosis or a bone related canceror metastases (e.g., malignancy metastatic to bone or multiple myeloma)and being treated with an active BP. In some embodiments, where asubject is not a candidate for a drug holiday and is at risk fordeveloping BRONJ and/or bisphosphonate-related symptoms as a result ofbeing treated with an active N-BP, one or more inactive BPs may beapplied locally to the site where the active N-BP is to be removed ordisplaced.

In embodiments where etidronate is administered to a subject as theinactive BP, etidronate is locally administered to the site where theactive N-BP is to be displaced or removed. In some embodiments,etidronate is locally administered as a preventative measure against thedevelopment of BRONJ and/or bisphosphonate-related symptoms.

BRONJ Preventive/Therapeutic Modality: Disclosed herein is the firstpreventive and/or therapeutic procedure for BRONJ based on displacementof active BPs with inactive BPs. As disclosed herein, the administrationof one or more inactive BPs likely results in a “competitiveequilibrium”-based displacement of active BPs. In the context of BPadsorption to bone, “BP displacement” refers to the displacement ofpreviously adsorbed BP with a subsequently administered BP. BPdisplacement has been demonstrated in vitro (FIG. 1) and in vivo (FIG.10, FIG. 12A-FIG. 12C). As disclosed herein, BP displacement may be usedas a preventive modality of BRONJ. For example, pre-adsorbed active BPmay be effectively displaced by one or more inactive BPs that isadministered systemically by, for example, intravenous or oraladministration, or by local administration to a given treatment site(e.g., direct intraoral injection to the jaw or topical application tothe oral mucosa).

Intraoral Approach: Local administration of one or more inactive BPs tothe site of a dentoalveolar procedure such as a tooth extraction candisplace one or more active BPs present in the bone, and thereby treator reduce the risk of developing BRONJ. Importantly, intraoralapplication of one or more inactive BPs to the site of a dentoalveolarprocedure, such as a tooth extraction can displace one or more activeBPs present at the site and thereby reduce a subject's risk ofdeveloping BRONJ without significantly interfering with the therapeuticactivity of active BPs in other tissues and/or at other sites within thesubject. Thus, in some embodiments, a pre-adsorbed active BP may beeffectively displaced or removed by intraoral injection of one or moreinactive BPs to the jaw. In some embodiments, one or more inactive BPsmay be administered in the form of a solution, gel or paste applied tothe site to be treated. As an example of a treatment method according tothe present invention, a subject may be administered an effective amountof one or more inactive BPs by intraoral application to the site of adentoalveolar procedure, e.g., a tooth extraction, to treat or inhibitBRONJ and/or bisphosphonate-related symptoms or reduce the risk of thesubject developing BRONJ and/or bisphosphonate-related symptoms. In someembodiments, one or more inactive BPs is applied to gingival/palataltissue of a subject before, during, and/or after a dentoalveolarprocedure.

Fluorescently Labeled Bisphosphonates (FL-BP): Inactive BPs can begenerated by conjugation of an active BP, such as RIS or ZOL and relatedanalogues, with a detectable label, e.g., a fluorescent compound.Examples of suitable fluorescent compounds include fluorescein andderivatives thereof (including carboxyfluorescein (FAM),Carboxyl-X-Rhodamine (ROX), Alexa Fluor 647 (AF647), Rhodamine Red-X(RhR-X), IRDye 800CW (800CW), Sulfo-Cy5, indocyanine green (ICG), andanalogues thereof, including those employed in the synthetic methods ofFIG. 18A1 to FIG. 18A4, and those depicted in FIG. 18B. Examples ofsuitable FL-BPs are disclosed in FIG. 2A-FIG. 2D, FIG. 5A, FIG. 5B, FIG.6, FIG. 18A1-FIG. 18A4, and FIG. 18B. FL-BPs may be used to monitorbisphosphonate localization and interactions in vivo, including bydirect visualization of osteoclast incorporation of a bisphosphonateadsorbed onto the bone mineral surface (FIG. 3). FL-BPs, which areinactive BPs and include ICG-BPs, may be used to remove or displaceactive BPs in skeletal tissue as disclosed herein.

One or more inactive BPs with different mineral binding affinities,antiresorptive activities (ranging from inactive to partially active),and/or functions may be employed. For example, a series of differentFL-BPs may be used to characterize the extent and amount of adsorbed insubjects, e.g., animal models, or monitor such treatments.

A feature of modern BPs such as RIS or ZOL is that their anti-resorptiveeffect depends on the structure of their nitrogen-containingsubstituent, distinct from their avid bone affinity, which is primarilydue to the two phosphonate groups. In some embodiments, the inactive BPsaccording to the present invention have scaffolds that are based onN-containing bisphosphonates, e.g., risedronate (RIS) and zoledronate(ZOL), exhibit bone affinity that is the same or substantially similarto N-containing bisphosphonates yet exhibit little to noanti-osteoclastic activity. In some embodiments, the inactive BPsaccording to the present invention have scaffolds that lack an α-hydroxygroup, a pyridyl group that is para-substituted (FIG. 4), or both, whichwill dramatically decrease antiresorptive activity without a significantimpact on bone affinity as compared to the corresponding active BPs. Insome embodiments, the inactive BPs according to the present inventioncomprise an active BP conjugated to a fluorescent compound, wherebyconjugation to the fluorescent compound renders the bisphosphonateinactive or less active as an antiresorptive agent. These fluorescentlylabeled bisphosphonates (FL-BPs) are capable of being strongly adsorbedto the surface of hydroxyapatite, but substantially or entirely lack theanti-resorptive activity of the corresponding unconjugatedbisphosphonate.

In some embodiments, the subject to be treated is an animal model. Insome embodiments, the subject to be treated is a human. In someembodiments, the subject to be treated is at risk of developing BRONJ.In some embodiments, the subject to be treated has been treated with oneor more active BPs.

In some embodiments, the present invention is directed to displacing apre-adsorbed active BP drug in a bone of a subject which comprisesadministering one or more inactive BPs to the bone of the subject.

In some embodiments, the amount of one or more inactive BPs administeredto the subject is a therapeutically effective amount or an effectiveamount. As used herein, an “effective amount” is a dose that results inan observable difference as compared to a placebo. In some embodiments,an effective amount of one or more inactive BPs is one that displacesmore than 50% of an active BP in a tissue, such as bone, whenadministered thereto. A “therapeutically effective amount”, refers to anamount of one or more compounds of the present invention that, whenadministered to a subject, (i) treats or inhibits a particular disease,condition, or disorder, (ii) attenuates, ameliorates, or eliminates oneor more symptoms of the particular disease, condition, or disorder,and/or (iii) inhibits or delays the onset of one or more symptoms of theparticular disease, condition, or disorder, as compared to a control. Atherapeutically effective amount of one or more compounds of the presentinvention will vary depending upon factors such as the givencompound(s), the pharmaceutical formulation, route of administration,the type of disease or disorder, the degree of the disease or disorder,and the identity of the subject being treated, but can nevertheless bereadily determined by one skilled in the art. For example, a“therapeutically effective amount” of one or more inactive BPs is onethat treats, inhibits, prevents, or reduces a sign or symptom of BRONJas compared to a negative control. Therapeutically effective amounts maybe determined from animal models. For example, a therapeuticallyeffective amount for a human can be formulated based on amounts thathave been found to be therapeutically effective in animal models.

In some embodiments, a therapeutically effective amount of the one ormore inactive BPs are administered as a single dose of about 5-20, about10-15, or about 11-12 milligrams per kilogram weight of the subjectprior to, during, or after an event, such as a dentoalveolar procedure,tooth extraction, implant procedures, the establishment ofperiodontitis, and the like. In some embodiments, the one or moreinactive BPs are administered during or immediately after the event. Theskilled artisan will appreciate that certain factors may influence thedosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent.

In some embodiments, the present invention is directed to a compositioncomprising, consisting essentially of, or consisting of one or moreinactive BPs. Compositions of the present invention, includingpharmaceutical compositions, comprise one or more inactive BPs and apharmaceutically acceptable carrier. As used herein, the terms“pharmaceutical composition” and “pharmaceutical formulation” are usedinterchangeably to refer to a composition suitable for pharmaceuticaluse in a subject. A pharmaceutical composition generally comprises anactive agent, e.g., one or more inactive BPs and a pharmaceuticallyacceptable carrier, e.g., a buffer, adjuvant, diluent, and the like.

The one or more inactive BPs to be administered to a subject may beprovided as a pharmaceutical formulation. Pharmaceutical formulationsmay be prepared in a unit-dosage form appropriate for the desired modeof administration. The pharmaceutical formulations of the presentinvention may be administered by any suitable route including oral,rectal, nasal, topical (including buccal and sublingual), vaginal,mucosal, and parenteral (including subcutaneous, intramuscular,intravenous, and intradermal). It will be appreciated that the route ofadministration may vary with the condition and age of the recipient, thenature of the condition to be treated, and the given compound(s) of thepresent invention. In some embodiments, the route of administration isoral. In some embodiments, the route of administration is mucosal. Insome embodiments, the one or more inactive BPs are delivered to the oralmucosa of a subject. In some embodiments, a pharmaceutical formulationaccording to the present invention is an aqueous solution of the one ormore inactive BPs. In some embodiments, a pharmaceutical formulationaccording to the present invention is a suspension, e.g., in alipophilic gel, comprising one or more inactive BPs. In someembodiments, a pharmaceutical formulation according to the presentinvention is a liposomal or nanovesicle preparation comprising one ormore inactive BPs encapsulated in the liposomes or nanovesicles. In someembodiments, the nanovesicles are nondeformable nanovesicles. In someembodiments, the nanovesicles are deformable nanovesicles. See, e.g., WO2017/087685 and Subbiah, et al. (2017) J Drug Delivery, Article ID4759839. In some embodiments, a pharmaceutical formulation according tothe present invention is a trans-oral mucosal gel formulation comprisingone or more inactive BPs. In some embodiments, a pharmaceuticalformulation according to the present invention is an oro-dentalmucoadhesive proniosomal gel formulation comprising one or more inactiveBPs.

It will be appreciated that the actual dosages of the inactive BPs usedin the pharmaceutical formulations will vary according to the particularcompound(s) being used, the particular composition formulated, the modeof administration, and the particular site, subject, and disease beingtreated. Optimal dosages for a given set of conditions may beascertained by those skilled in the art using dosage determination testsin view of the experimental data for a given compound. Administration ofprodrugs may be dosed at weight levels that are chemically equivalent tothe weight levels of the fully active forms.

Pharmaceutical compositions and formulations according to the presentinvention comprise a therapeutically effective amount of one or moreinactive BPs and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable vehicle” and “pharmaceutically acceptablecarrier” are used interchangeably and refer to and include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, excipients, diluents,and the like, that are compatible with pharmaceutical administration andcomply with applicable standards and regulations, e.g., thepharmacopeial standards set forth in the United States Pharmacopeia andthe National Formulary (USP-NF) book, for pharmaceutical administration.Thus, for example, unsterile water is excluded as a pharmaceuticallyacceptable carrier for, at least, intravenous administration.Pharmaceutically acceptable vehicles include those known in the art.See, e.g., Remington: The Science and Practice of Pharmacy. 20^(th) ed.(2000) Lippincott Williams & Wilkins. Baltimore, Md., which is hereinincorporated by reference. The pharmaceutically acceptable carrieremployed may be either a solid or liquid. Exemplary of solid carriersare lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesiumstearate, stearic acid, and the like. Exemplary of liquid carriers aresyrup, peanut oil, olive oil, pharmaceutical grade water, and the like.Similarly, the carrier or diluent may include time-delay or time-releasematerial known in the art, such as glyceryl monostearate or glyceryldistearate alone or with a wax, ethylcellulose,hydroxypropylmethylcellulose, methylmethacrylate, and the like. In someembodiments, the pharmaceutically acceptable carrier is a liposome. Insome embodiments, the pharmaceutically acceptable carrier is ananovesicle, which may be deformable or nondeformable. In someembodiments, the pharmaceutically acceptable carrier is a proniosome.

Toxicity and therapeutic efficacy of inactive BPs can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds exhibiting large therapeutic indices are preferred. Whilecompounds that exhibit toxic side-effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, a therapeutically effective dose can beestimated initially from cell culture assays. A dose may be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC₅₀ (i.e., the concentration of the test compoundthat achieves a half-maximal inhibition of symptoms) as determined incell culture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by high performance liquid chromatography.

EXAMPLES

The following examples are intended to illustrate but not to limit theinvention.

Animals Mouse Osteonecrosis of the Jaw (ONJ) Model

The C57BL/6J (B6) mice model of ONJ is used. The consistent tissuesource in this study is mouse maxillary bone with or without toothextraction, as well as femurs, mandibles and L4/L5 lumber bones. Toothextraction animal models for studying wound healing in mice have beenwell established in our and other laboratories. Systemic bisphosphonatetreatment is accomplished by IV injection and the proposed localapplication of FL-BP is accomplished by intraoral injection togingival/palatal tissue of mouse maxilla. Mice are subjected tomaxillary molar extraction. After anesthesia and vital signs checked,the animal is placed on a bed supporting the isoflurane tubing whilekeeping the mouth opening. Ophthalmic ointment (bland, e.g., Lacrilube)is applied. The maxillary molar area is wiped with 0.12% Chlorhexidinegluconate solution (Peridex, oral rinse). An autoclaved dental exploreris used as an exodontia elevator after careful reflection of thecervical mucosa to minimize post-extraction discomfort. An autoclavedcotton swab is placed on the extraction socket until the initial bloodcoagulation is established. Maxillary 1st molar is then extractedunilaterally from so that the remaining untreated side can be utilizedfor chewing. In our previous experiments, the entire procedure takesabout 5-15 minutes.

ONJ occurs in the oral cavity. Dental procedures (commonly toothextraction) have been noted as a trigger for the onset ofbisphosphonate-associated ONJ. It is necessary to use animals to performthe proposed research because there are no appropriate alternativemodels of oral wound healing. It would be impossible to study thebiology and molecular mechanism of wound healing if other models areused. Extrapolation of the future wound healing treatment to the humanpatients would be impossible without using animals. Mice are usedbecause their similarity in wound healing process to humans; and theimmediate availability of genetically modified strains that are directlyrelevant to our hypothesis. The estimated number of animals required forthis study is based on the consideration that a comprehensive study ofbisphosphonate treatment on wound healing will be achieved.

Veterinary care is provided through the Department of Laboratory AnimalMedicine at UCLA Medical Center. Animal subjects are examined followingsurgery; veterinary consultation is obtained for complications such asinfection. No animals are used which shows signs of respiratory diseaseor distress, or enteric disease. UCLA is fully accredited the AmericanAssociation of Accreditation of Laboratory Animal Care (AAALAC).

To prevent any distress, discomfort, or pain to any of the animals,procedures are performed under general anesthesia, using isofluraneinhalation (1-2%). After the survival surgery, the animal is placed on awater-circulating heating pad. The animal is monitored forcardiovascular and respiratory function and attended continuously untilthe animal forms a sternal position. To prevent post-proceduraldiscomfort, analgesics are administered. Whereas no special treatmentsare considered for the husbandry of these mutant mice, any possibleinfections are monitored.

Animals are euthanized at the completion of experiments. Euthanasia iscompleted by 100% carbon dioxide inhalation. The method is consistentwith the recommendations of the Panel of Euthanasia of the AmericanVeterinary Medical Association.

Example 1

Synthesis and Characterization of FL-BPs with Variable Mineral BindingAffinities

Bisphosphonates having a fluorescent compound conjugated thereto(FL-BPs) that have low or undetectable biological activity but possesshigh bone mineral affinity were synthesized. Different fluorophores wereconjugated to bisphosphonates using “Magic linker” technology (see,e.g., U.S. Pat. No. 8,431,714). In vitro characterization of bindingaffinity/pharmacological activity was performed.

Experiment 1-1: Synthesis of FL-BPs

FL-BPs were synthesized using “Magic linker” technology (see, e.g., U.S.Pat. No. 8,431,714) to conjugate BP compounds, e.g., RIS, ZOL, and otherrelated analogues (e.g., FIG. 4) to fluorophores as schematically shownin FIG. 5A. The conjugates, FL-BPs, were obtained in 50-77% yield (>98%purity) and were characterized by UV-VIS, fluorescence emission, 1H NMR,31P NMR, and HRMS. FIG. 6 shows the structural formulas of three FL-BPsthat were synthesized. In some embodiments, BPs used according to thepresent invention are those as set forth in U.S. Pat. No. 8,431,714,which is herein incorporated by reference in its entirety. An example ofan low activity BP is 1-hydroxy-2-(pyridin-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS); another example of an inactive BP is2-(pyridin-4-yl)ethane-1,1-diyl bisphosphonic acid (p-PyrEBP).

Experiment 1-2: In Vitro Characterization of FL-BPs A) Mineral BindingAffinity Assay

Hydroxyapatite assays indicate that the FL-BPs generally retainsubstantial affinity for bone mineral that reflect the affinity of theBP component and to a lesser extent, the affinity of the conjugatedfluorophore. Hydroxyapatite (Macro-Prep® Ceramic hydroxyapatite Type II20 μM) was obtained from Bio-Rad Laboratories, Inc. Hercules, Calif.Accurately weighed hydroxyapatite powder (1.4-1.6 mg) was suspended in a4 mL clear vial containing the appropriate volume of assay buffer (0.05%(wt/vol) Tween20, 10 μM EDTA and 100 mM HEPES pH=7.4) for 3 hours.Hydroxyapatite was then incubated with increasing amounts of FAM-BP andROX-BP (0, 25, 50, 100, 200, and 300 μM). Samples were gently shaken for3 hours at 37° C. in appropriate volume of assay buffer (0.05% (wt/vol)Tween20, 10 μM EDTA and 100 mM HEPES pH=7.4). Subsequent to theequilibrium period, the vials were centrifuged at 10,000 rpm for 5minutes to separate the solids and the supernatant. 0.3 mL of thesupernatant was collected and the equilibrium solution concentration ismeasured by using Shimadzu UV-VIS spectrometer. For the calibrationseries, FL-BP probe standards were prepared by serial dilution from thestock solution with the same isotherm buffer to give the range from 0 to400 μM. Calibration curves were constructed using standard solutions ofthe target FL-BPs. Fluorescent emission can also be used to calculatethe binding parameters. Nonspecific binding was measured with a similarprocedure in the absence of hydroxyapatite as control. Bindingparameters (Kd and Bmax, represent the equilibrium dissociate constantand maximum number of binding sites, respectively) were calculated usingthe PRISM program (Graphpad, USA). The binding parameters of eachcompound was measured in 5 independent experiments. The compounds withequilibrium dissociation constant (Kd) higher than about 100 μM (about10× of Kd of parent BPs) were eliminated from further investigation. Atwo-sample t-test was used to evaluate the binding parameters of theprobes. The sample size (n=5) in each group was able to detect theeffect size 1.72 for this hypothesis at a power of 80% and a one-sideType I error of 0.05.

Hydroxyapatite assays confirm that the FL-BPs generally retainsubstantial bone mineral affinity. The fluorophore component does exertsome influence on affinity: 5-FAM-ZOL and AF647-ZOL showed a smallreduction, while ROX-RIS showed an enhancement (FIG. 7). Thus, the boneaffinity of bisphosphonates is substantially retained when conjugated todifferent fluorophores.

A method based on Langmuir adsorption isotherms was used to measurebinding affinity measurement of representative fluorescent BP probes;the results are in good agreement with results from the hydroxyapatitecolumn assay (FIG. 8A-FIG. 8D).

B) Biological Activity In Vitro Assay

In vitro activity assays were performed by using bone marrow macrophagesand culturing them with M-CSF on bovine bone slices for 2 days to enrichfor osteoclast precursors (OCPs) and then treating with RANKL for afurther 3 days to develop and isolate multinucleated osteoclasts. Boneslices were pre-adsorbed with FL-BPs and serially diluted numbers ofOCPs were cultured on them. Resorption pit areas were measured.Separately, osteoclasts were collected and total protein samples wereprepared. The prenylation of Rapla were examined by Western blot. Eachexperimental group (n=5) will be compared with the control (n=5) in theprototype experiment. Experiment 1-2-B has the same power calculation asExperiment 1-2-A.

In a preliminary study, to determine the effect of fluorescent ZOLanalogues on cell viability, J774.2 mouse macrophages were plated at2×10⁵ cells/ml in 96-well plates and left to adhere overnight. Cellswere then treated with 10, 100, or 500 μM of fluorescent ZOL analogues,ZOL, or vehicle, for 48 hours at the end of the incubation period, themedium was removed, and cells were washed twice with PBS. Medium withAlamarBlue reagent was then added and cells are incubated for a further3 hours. Western blot and cell viability assays indicated a significantbiological effect of the FAM conjugates. These results demonstrate that5-fluorescein-ZOL conjugates (and similar RIS conjugates) retainobservable antiprenylation and the anti-resorptive properties of theparent BP drug.

Alternative Approaches

Although most of the in vitro systems and assays are well establishedfor fluorescent BP probes, hydroxyapatite column assays may be used forbinding affinity assays. Dentine discs may also be used instead ofbovine bone slices. The methodology can be applied to generate otherFL-BPs with appropriate modifications on the structure of BP molecules,such as p-RIS, to obtain the desired affinity.

Example 2 Oral Application of FL-BP for Local Adsorption to Jawbone andCompetitive Displacement of Pre Adsorbed FL-BP Via IV Injection

This experiment was carried out to demonstrate BP displacement in vivo.Pre-adsorbed FL-BP in mice resulting from systemic administration viatail vain injection is challenged by a local application togingival/palatal tissue of maxilla using a FL-BP carrying differentfluorophore. Local application may include: a) injection togingival/palatal tissue; b) mouth wash; and c) topical application fortrans-mucosal delivery. FL-BP may be applied to the periosteal space ofjawbone. The dose and the time point of oral injection is established byquantitative fluorescence imaging analysis to achieve maximumdisplacement efficiency. Consideration was also given to possibledistant toxic effects. Femurs, mandibles, and L4/L5 lumber bones wereexamined.

Experiment 2-1

This experiment establishes the oral injection protocol and determinesthe relationship between injection doses and maxillary bone adsorptionfluorescent signal. A total of 30 female B6 mice are divided in 5 groups(n=6). In each group, a single intraoral injection of 0, 1, 5, 10, or 50μM of ROX-BP in 25 μl of 0.9% NaCl solution were performed to thegingival/palatal tissue. One week after the oral injection, skull(containing the maxillary injection site), mandible, femur, and L4/L5lumber bones were harvested and soft tissues were removed. Bonespecimens kept in PBS were evaluated by the standardized fluorescentbiophotonics and the signal was quantified using the proprietary program(LAS3000, FUJIFILM).

Experimental Results

As shown in FIG. 9, ROX and FAM have overlapping emission spectraresulting in suboptimal fluorescent signal separation (data not shown).Therefore, AF647-ZOL was selected as the candidate FL-BP to testdisplacement of FAM-ZOL and vice versa. Use of complementary fluorescentsignals from the original and displacing labeled BP permitted theoriginal BP loading and its response to the displacing BP loading to bemonitored.

The mouse intra-oral injection protocol was established by using aHamilton syringe with a 33-gauge needle. Under a surgical microscope, 2μl solution was injected to the palatal gingiva of left first molar ofmouse maxilla. After slow injection, the needle was left in the tissuefor 3 minutes and then gently removed.

Using this method, 2 μl of 0 μM, 0.5 μM or 5.0 μM AF647-ZOL was injectedto mouse maxillary oral mucosa to mice 6 days after retro-orbital IVinjection of 100 μl of 50 μM FAM-ZOL. Mouse tissues were harvested 1 dayafter oral injection and maxillary bones were subjected to standardizedfluorescent biophotonics image obtained using an excitation wavelengthof 460 nm and a 515 nm filter (LAS3000, FUJIFILM Corp).

As shown in FIG. 10, this method consistently and successfully deliveredFL-BP to mouse maxillary oral mucosa tissue. AF647-ZOL intra-oralinjection gave rise to a dose dependent AF647 signal at and around theinjection site on the palatal bone. Furthermore, there was an indicationthat pre-adsorbed FAM-ZOL was removed by the AF647-ZOL. However, thequantitative evaluation of this displacement presented some difficulty,largely due to the complex anatomy of mouse maxilla.

Experiment 2-2

This experiment examines the postulated BP displacement at the intraoralinjection site. A total of 30 female mice were divided into 5 groups(n=6). All groups received a single IV injection of 200 μl of 350 μMFAM-BP. One week later, mice in each group received an intraoralinjection of 25 μl of 0, 1, 5, 10, or 50 μM ROX-BP to gingival/palataltissue. Mice were euthanized 1 week after the intraoral injection andskull, mandible, femur and L4/L5 lumber bone were harvested. The bonespecimens were evaluated for 2 color fluorescent biophotonics and eachsignal was quantified.

In a preliminary study female C57Bl6/J (B6) mice (8 weeks old) receivedeither a single IV injection via tail vein of 200 μl of 350 μM 5-FAM-ZOL(5-FAM-ZOL, #BV111001, BioVinc LLC, Culver City, Calif.) or a singleintraoral injection of 25 μl of 50 μM ROX-RIS (5(6)-ROX-RIS, #BV150101,BioVinc LLC, CA) to gingival/palatal tissue at maxillary left firstmolar (white arrow in FIG. 11). One week later, mice were euthanized 3weeks after the IV injection of 5-FAM-ZOL or 2 days after the intraoralinjection of ROX-RIS. Skulls (containing maxilla) were harvested andstandardized fluorescent biophotonics image was obtained using anexcitation wavelength of 460 nm and the 515 nm filter (LAS3000, FUJIFILMCorp). Strong and relatively uniform green fluorescent signal wasdetected throughout the maxilla and zygomatic arch after the IVinjection of FAM-ZOL. By contrast, the palatal bone around the intraoralinjection site of ROX-RIS showed a localized red fluorescent signal(FIG. 11).

Experimental Results

Mice were pre-treated by 100 μl of 50 μM AF647-ZOL retro-orbital IVinjection. Six days later, mice received sub-periosteal injection tocranial bone with 10 μl of 0 μM, 1 μM, 10 μM, or 50 μM FAM-ZOL. Thecranial bone was harvested and soft tissues were removed. Thefluorescent signals of AF647 and FAM were measured by standardizedfluorescent biophotonics. The cranial bones were then prepared forcryosection. Cross-sections were made by the tape method and evaluatedby confocal laser scanning microscopy.

The FAM signal of sub-periosteally injected FAM-ZOL showed a dosedependent increase on the cranial bone at the injection site. As shownin FIG. 12A, AF647 signal at the corresponding site showed a gradualdecrease with the increasing dose of FAM-ZOL. Statistical significancewas reached at 10 μM and 50 μM as shown in FIG. 12B.

The cranial bone cryo-cross sections revealed AF647-ZOL labeling on theexternal and internal bone surfaces, as well as trabecular surface asshown in FIG. 12C. In the cranial bone specimen received FAM-ZOLsub-periosteal injection, a FAM-ZOL signal was observed primarily on theexternal bone surface and, occasionally, in the bone marrow space. TheAF647-ZOL signal on the external bone surface was disproportionatelyreduced as compared to the internal bone surface (FIG. 12C).

Alternative Approaches

If desired, the data from the above study could be used to perform poweranalysis and adjust the number of animals. In addition to the proposedfluorescent biophotonics imaging method, one may use non-decalcifiedbone cryosection, which may provide the depth of adsorbed FL-BP in bone.Alternatively, bone pieces may be sampled and decalcified in vitro torelease the adsorbed FL-BP. The released FL-BP may be quantified by2-color-plate reader or HPLC.

Example 3 BP Displacement Therapy for the Prevention of BRONJ In Vivo

This study examines the effectiveness of the proposed BP displacement inpreventing the development of BRONJ. The intraoral administration of aninactive and/or low activity FL-BP prior to tooth extraction was appliedto the established ZOL-induced mouse BRONJ model. The postulated diseasemodification was determined by the prevalence of BRONJ, as well ashistological oral mucosa abnormality and osteonecrosis development. Thisstudy establishes proof-of-concept for the proposed BP displacementtherapy.

Experiment 3-1

This study determines the anti-resorptive function of FL-BP in vivo, ascompared to ZOL. Female B6 mice were injected with 50 μM ZOL (n=6), 50μM FL-BP (n=6) or 0.9% NaCl vehicle solution (control: n=6) via tailvein. Three weeks later, mice were euthanized and femur and L4/L5 wereharvested. After fixed in 10% buffered formalin, bone samples weresubjected to micro CT analysis. The anti-resorptive function werequantitatively established by the trabecular bone three-dimensionalmorphometry (e.g., BV/TV, Tb.N, Tb.Th, TbSp). 5-FAM-ZOL developed byBioVinc exhibited pharmacological function as shown in FIG. 13. FL-BPswith minimal or no pharmacological BP function are used. As used herein,a compound that has minimal or no pharmacological BP function means thatthe compound exhibits little to no anti-resorptive activity as measuredby a protein anti-prenylation assay (see Kashemirov (2008) BioconjugateChem. 19(12): 2308-2310; Sun (2016) Bioconjugate Chem. 27(2): 329-340).

Experimental Results

Mice were IV injected with 0.9% NaCl (vehicle solution), ZOL (184μM×100=5.0 μg/animal) or AF647-ZOL (184 μM×100 μL=22.0 μg/animal) andfemurs were harvested 2 weeks after injection. The micro CT data werecompared.

ZOL injection nearly doubled the volume of femur trabecular bone (BV/TV)and the connectivity density. By contrast, as shown in FIG. 14,AF647-ZOL did not change any of bone structural parameters. This studysuggested that AF647-ZOL did not have the antiresorptive pharmacologicaleffect of ZOL.

Experiment 3-2

Female B6 mice were treated with 200 μl of 350 μM ZOL IV injection viatail vein. The ZOL dose for mice was calculated by metabolic scaling ofhuman dose for cancer patient. One week later, the intraoral injectionFL-BP was performed at the maxillary first molar area ofgingival/palatal tissue. FL-BP used in this experiment possessedequivalent bone affinity to ZOL; but significantly reduced BP function.One week after the intraoral injection, the maxillary left first molarwas extraction. Mice in each group were euthanized at week 2 (n=6 ineach group) and at week 4 (n=6 in each group). Maxillary tissueincluding the tooth extraction site, mandible, femur, and L4/L5 lumberbones were harvested. All bone samples were fixed in 10% bufferedformalin and analyzed by micro CT. After decalcification by 10% EDTA,paraffin sections were used for histopathological examination. Thesections with cytokeratin 14 immunohistology were examined forepithelial pathology and TRAP for osteoclast.

In a preliminary study female B6 mice were injected 500 μg/Kg ZOL(equivalent to 200 μl of 350 μM ZOL) via tail vein and one week later,maxillary left first molar was extracted. Tooth extraction wound ofcontrol mice injected with vehicle solution was closed in 2 to 4 weeks,whereas 100% and 50% of ZOL-injected mice showed open oral wound at 2weeks and 4 weeks of tooth extraction, respectively (FIG. 15).

Experimental Results

Mice received a bolus IV injection of ZOL (440 μg/Kg; 294 μM×100 μL=8.0μg/animal) from retro-orbital venous plexus. As shown in FIG. 16A,eleven (11) days later, mice received AF647-ZOL intra-oral injection(250 μM×2 μL=0.59 μg/animal) or AF647-ZOL IV injection (500 μM×100μL=59.2 μg/animal) and then one day after AF647-ZOL injection, mice weresubjected to extraction of the left maxillary first molar and the oralwound healing was evaluated 2 weeks after tooth extraction. TheAF647-ZOL signal in distant bone was assessed in femurs.

FIG. 16B shows that ZOL-injected disease control mice showed delayedwound healing with gingival swelling and jawbone exposure, consistentwith the development of a BRONJ-like lesion. Intra-oral injection ofAF647-ZOL 1 day before the tooth extraction resulted in the completeprevention of BRONJ without oral mucosa inflammation/swelling in allindividuals (n=4) in this group. By contrast, some mice which receivedan AF647-ZOL IV injection showed signs of oral mucosainflammation/swelling, although jawbone exposure was much attenuated(FIG. 16B). Evaluation of femurs demonstrated a clear AF647-ZOL signalin mice which received AF647-ZOL by IV injection but not in mice whichreceived AF647-ZOL by intra-oral injection as shown in FIG. 16C. Theoutcome indicated that intra-oral application and localized BPdisplacement in jawbone is advantageous.

Alternative Approaches

Human ONJ cases are determined after 8 weeks of non-healing oral wound.Because mouse exhibits faster metabolism and accelerated wound healing,this study can observe tooth extraction wound for 4 weeks. By this time,control mice show complete wound healing. However, the effect of theFL-BP therapeutic intervention may need to be monitored for a longerperiod.

The results for the experiments with AF647-ZOL indicate that 1)Equilibrium-based BP displacement occurs in vivo, and thereby supportsthe therapeutic use of inactive BPs for preventing, inhibiting, and/ortreating BRONJ, 2) fluorescent compounds, such as AF647, may beconjugated to a BP, and when conjugated thereto, the antiresorptiveactivity of the BP is reduced to a minimum, while the mineral bindingefficiency is retained, and 3) intra-oral application of inactive BPs iseffective in preventing, inhibiting, or treating BRONJ.

Example 4 Alkyl Bisphosphonates

Alkyl bisphosphonates such as methylene hydroxyl bisphosphonate (MHDP orMHBP), ethylene hydroxyl bisphosphonate (EHDP), methylene bisphosphonate(MBP), clodronate, and the like (FIG. 17) may displace N-BPs insufficient amounts to prevent, reduce, or inhibit the deleterious actionof N-BPs in the jaw and other skeletal sites. The alkyl BPs can have afluorescent compound conjugated thereto, some of which are illustratedin FIG. 18.

The results shown in FIG. 19A document the lack of anti-resorptiveactivity of MHDP, thereby indicating that such alkyl BPs can be used totreat, inhibit, reduce, or prevent BRONJ and other bone and skeletalproblems caused by active BPs. Western blot analysis confirmed theseresults and indicated that ZOL but not MHDP and ETI (etidronate)inhibited Rap1A prenylation (accumulated unprenylated Rap1A (uRap1A)) inJ774 macrophages at 10 μM and 100 μM dose.

Therefore, as schematically shown in FIG. 19B mouse maxillae weretreated with ZOL and then either 2 μg of MHDP was orally injected to thesite of the molar to be extracted or 100m of MHDP was IV injected andthe sites of molar extraction of treated mouse maxilla were compared tocontrols. As shown in FIG. 19C, intra-oral injection and IV injection ofMHDP prevented the development of ONJ-like lesions as compared to theuntreated controls.

Example 5 Near Infrared FL-BPs

Near infrared (NIR) FL-BPs were synthesized as novel theranostic agents(FIG. 18): specifically, the indocyanine green-BP (ICG-BP) conjugates:the fluorophore ICG attached to p-PyrEBP or p-RIS (FIG. 4). Unlike thepotent antiresorptive N-BPs RIS or ZOL, the BP scaffolds of p-PyrEBP andp-RIS either lack an α-hydroxy group and/or its pyridyl side chain arebe para-substituted, which will decrease antiresorptive activity to anegligible level, with only a minor effect on bone affinity. The designthus allows the FL-BP conjugates to be strongly adsorbed to the surfaceof hydroxyapatite, while not exhibiting the biological activities of theparent N-BPs.

ICG, the only clinically approved NIR fluorescent dye for human use,absorbs around 800 nm and fluoresces in the NIR region (600-1200 nm)with high fluorescence intensity, and has been used widely in imagingstudies involving the heart, liver, lungs, blood circulation, and lymphnodes. An imaging system (Xiralite X4, Mivenion GmbH, Berlin, Germany)has recently been approved for use in the US, and the hand-heldinstruments (e.g., KaVo DIAGNOcam) are currently being used to identifycaries in teeth using NIR wavelength light, which are readily adaptableto the ICG-BPs here. Thus, this novel composition of matter allows easeof visualization of BPs without antiresorptive effects in bone.

However, ICG does not possess any native functionality that would permitlinkages. Thus, a carboxyl moiety was introduced into the molecule, insuch a way that conjugation with a BP is possible at three alternativepositions. These scaffolds are designed to have minimal effect on thefluorophore core, preserving the optical properties of the ICGfluorophore (FIG. 18).

These novel ICG-BPs can be used to allow safe dental procedures to becarried out in any patient undergoing BP therapy for osteoporosis,including highly dosed N-BPs in cancer associated bone disease. Ideally,the product can lead to minimal systemic adsorption of the FL-BP, whileallowing a dentist to use an inexpensive hand-held light to visiblyascertain effective dosing at the jawbone site and then initiate thedental procedure. The use of NIR fluorescent conjugates is a keyinnovation because it allows the visualization of the maxillofacial bonethrough the overlaying fascia and tissue to allow monitoring for patientvariation in conjugate uptake, such as might be due to differentialjawbone turnover levels depending on such individual patentcharacteristics age, gender, ethnicity (personalized or precisionmedicine). This improves ease of use and increases cost effectiveness,while increasing the confidence of the dentist in the outcome of theprocedure, due to the ability to observe directly the loading of thetheranostic agent.

REFERENCES

In addition to the references cited throughout, which are hereinincorporated by reference in their entirety, the following referencesare also incorporated herein by reference in their entirety:

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All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified.

As used herein, the terms “subject” and “patient” are usedinterchangeably and include humans and non-human animals. The term“non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, horses, sheep, dogs, cows,pigs, chickens, and other veterinary subjects and test animals.

The use of the singular can include the plural unless specificallystated otherwise. As used in the specification and the appended claims,the singular forms “a”, “an”, and “the” can include plural referentsunless the context clearly dictates otherwise. As used herein, “and/or”means “and” or “or”. For example, “A and/or B” means “A, B, or both Aand B” and “A, B, C, and/or D” means “A, B, C, D, or a combinationthereof” and said “combination thereof” means any subset of A, B, C, andD, for example, a single member subset (e.g., A or B or C or D), atwo-member subset (e.g., A and B; A and C; etc.), or a three-membersubset (e.g., A, B, and C; or A, B, and D; etc.), or all four members(e.g., A, B, C, and D).

The phrase “comprises, consists essentially of, or consists of” is usedas a tool to avoid excess page and translation fees and means that insome embodiments the given thing at issue comprises something, and insome embodiments the given thing at issue consists of something. Forexample, the sentence “In some embodiments, the composition comprises,consists essentially of, or consists of A” is to be interpreted as ifwritten as the following two separate sentences: “In some embodiments,the composition comprises A. In some embodiments, the compositionconsists essentially of A. In some embodiments, the composition consistsof A.” Similarly, a sentence reciting a string of alternates is to beinterpreted as if a string of sentences were provided such that eachgiven alternate was provided in a sentence by itself. For example, thesentence “In some embodiments, the composition comprises A, B, or C” isto be interpreted as if written as the following three separatesentences: “In some embodiments, the composition comprises A. In someembodiments, the composition comprises B. In some embodiments, thecomposition comprises C.” As used herein, the phrase “consistsessentially of” in the context of a composition comprising one or moreinactive BPs means the composition does not contain any active BPs, butmay contain other active ingredients, e.g., an analgesic, anantibacterial, etc.

To the extent necessary to understand or complete the disclosure of thepresent invention, all publications, patents, and patent applicationsmentioned herein are expressly incorporated by reference therein to thesame extent as though each were individually so incorporated.

Having thus described exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the within disclosuresare exemplary only and that various other alternatives, adaptations, andmodifications may be made within the scope of the present invention.Accordingly, the present invention is not limited to the specificembodiments as illustrated herein, but is only limited by the followingclaims.

1. A method of removing or displacing an active bisphosphonate in askeletal tissue, which comprises administering to the skeletal tissueone or more inactive bisphosphonates.
 2. The method according to claim1, wherein the one or more inactive bisphosphonates has a molecularscaffold that (a) lacks an α-hydroxy group, (b) has a pyridyl side chainthat is para-substituted, or (c) lacks an α-hydroxy group and has apyridyl side chain that is para-substituted.
 3. The method according toclaim 1, wherein the one or more inactive bisphosphonates has adetectable label attached thereto.
 4. The method according to claim 1,wherein the one or more inactive bisphosphonates comprises abisphosphonate conjugated to a fluorescent compound.
 5. The methodaccording to claim 4, wherein the fluorescent compound is selected fromthe group consisting of: ROX, FAM, AF647, ICG, ICG analogs, Cy5,Sulfo-Cy5, Cy7, and IRDye 800CW.
 6. The method according to claim 4,wherein the one or more inactive bisphosphonates is selected from thegroup consisting of: 5-FAM-dRIS, 5(6)-FAM-dRIS, 5(6)-FAM-RIS,5(6)-FAM-RISPC, 5(6)-RhR-RIS, 5(6)-RhR-dRIS, 5(6)-RhR-RISPC,5(6)-ROX-RIS, 5(6)-ROX-RISPC, 5-FAM-RIS, 5-FAM-ZOL, 6-FAM-RIS,800CW-ZOL, AF647-RIS, AF647-RISPC, AF647-ZOL, 800CW-RIS, 800CW-ZOL,800CW-RISPC, ICG-RIS, ICG-ZOL, ICG-RISPC, and ICG-p-pyrEBP.
 7. Themethod according to claim 4, wherein the one or more inactivebisphosphonates is 5(6)-ROX-RIS, 5-FAM-ZOL, AF647-ZOL, ICG-A-p-PyrEBP,ICG-B-p-PyrEBP, ICG-C-p-PyrEBP, ICG-D-p-PyrEBP, ICG-A-p-RIS, orICG-D-p-RIS.
 8. The method according to claim 1, wherein the activebisphosphonate is an active nitrogen-containing bisphosphonate.
 9. Themethod according to claim 8, wherein the active nitrogen-containingbisphosphonate is alendronate, ibandronate, minodronate, pamidronate,risedronate, or zoledronate.
 10. The method according to claim 1,wherein the active bisphosphonate in the skeletal tissue is removed ordisplaced in vivo in a subject, with the proviso that when the one ormore inactive bisphosphonates is etidronate, the mode of administrationis local, preferably topical.
 11. The method according to claim 10,wherein the one or more inactive bisphosphonates is locally administeredto the subject.
 12. The method according to claim 10, wherein the one ormore inactive bisphosphonates is administered orally to the subject. 13.The method according to claim 10, wherein the one or more inactivebisphosphonates is administered to a gingival tissue and/or a palataltissue of the subject.
 14. The method according to claim 10, wherein theone or more inactive bisphosphonates is administered by intraoralapplication to a site of a dentoalveolar procedure.
 15. The methodaccording to claim 14, wherein the one or more inactive bisphosphonatesis administered before, during, and/or after the dentoalveolarprocedure.
 16. The method according to claim 1, wherein an effectiveamount of the one or more inactive bisphosphonates is administered. 17.The method according to claim 1, wherein the one or more inactivebisphosphonates is administered in the form of a liposomal formulationor a nanovesicle formulation, preferably a deformable nanovesicleformulation.
 18. A method of treating, reducing, preventing, orinhibiting Bisphosphonate Related Osteonecrosis of the Jaw (BRONJ) or abisphosphonate-related symptom in a subject, which comprisesadministering to the subject a therapeutically effective amount of oneor more inactive bisphosphonates.
 19. The method according to claim 18,wherein the subject has been or is being treated with an activebisphosphonate.